Optimisation and Validation of a High Throughput S creening Compatible Assay to I dentify I nhibitors of the Plasma M embrane C alcium ATPase Pump - a N ovel Therapeutic T arget for Contraception and M alaria

Purpose . ATPases , which constitu te a major category of ion transporters in the human body , have a variety of significant biological and pathological roles. However, the lack of high throughput assays for ATPases has significantly limited drug discovery in this area. W e have recently foun d that the genetic deletion of the ATP dependent calcium pump PMCA4 (plasma membrane calcium/calmodulin dependent ATPase, isoform 4) result s in infertility in male mice due to a selective defect in sperm motility . In addition , recent discoveries in humans have indicated that a single nucleotide polymorphism (SNP) in the PMCA4 gene determine s the susceptibility towards malaria plasmodium infection. Therefore, there is an urgent need to develop specific PMCA4 inhibitors. In the current study, we aim to optimi se and validate a high throughput screening compatible assay using recombinantly expressed PMCA4 and the HTRF ® Transcreener ® ADP (TR - FRET ) assay to screen a drug library . Methods and Results. PMCA4 membrane microsomes were prepared from HEK293 cells overex pressing PMCA4. Western blot quantific ation revealed nearly nine - fold increased expression of PMCA4 compared to LacZ (contro l virus) - infected cells . Maximal PMCA4 microsomal activity was achieved in the TR - FRET assay with 15ng/ μl microsomal concentration, 30 - minute pre - incubation with compounds at 37°C, and calcium buffering with 1mM EGTA providing 1μM free - calcium. Finally a dose - response curve for carboxyeosin (a non - specific PMCA inhibitor) under optimised conditions showed s ignificant PMCA4 inhibition. Upon confirmation that the assay was suitable for high - throughput screening, we have screen ed the ChemBioNet small molecule library ( ~ 2 1 ,000 compounds ) against the PMCA4 assay to identify those that are its apparent inhibitors. This screening yielded 1 , 494 primary hits . Conclusions . We have optimi s ed the HTRF ® Transcreener ® ADP assay for high - throughput screening to identify PMCA4 inhibitors . The output of the screening campaign has provided preliminary chemical starting points that could be further developed to specific PMCA4 inhibitors for non - hormonal contraception or anti - malaria therapy . This article is open to POST - PUBLICATION REVIEW . Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.